Our laboratory focuses on elucidating the coupling of the forces, structure, and dynamics of biologically important macromolecules. The next challenge in structural biology is to understand the basis of the physical interactions between molecules in aqueous solution. The ability to take advantage of the increasing number of protein and nucleic acid structures determined by x-ray crystallography and solution NMR will depend critically on this knowledge in order to understand and predict the strength and specificity of interactions among biologically important macromolecules that control cellular function and to design rationally agents that can effectively compete with those specific interactions associated with disease. Our past results have shown that experimentally measured forces are very different from those expected from current, conventionally accepted theories of intermolecular interactions. We have interpreted the observed forces as indicating a dominating contribution from water structuring energetics. Our research program consists of two parts. We use osmotic stress and x-ray scattering to measure directly forces between biological macromolecules in macroscopic condensed arrays. In order to extend our investigations to the role of water in the interaction of individual molecules, we also measure and correlate changes in binding energies and in hydration accompanying specific recognition reactions of biologically important macromolecules in solution, particularly of sequence specific DNA-protein complexes. The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, the last 10-15 Angstroms separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, has led us to conclude that the energy associated with structuring water between close surfaces dominates intermolecular forces. We are currently focusing on understanding the connection between hydration force magnitudes and the chemical natures of the interacting surfaces. We have characterized the exclusion of alcohols from DNA using direct force measurements. Forces between DNA helices in macroscopic condensed arrays can be measured monitoring the distance between helices using x-ray diffraction as a function of the osmotic pressure of a polymer solution in equilibrium with the DNA phase. From the sensitivity of DNA forces to the concentration of alcohols or other solutes, the spatial distribution of alcohols or other solutes around DNA can be extracted. We find that repulsive hydration forces underlie the preferential hydration of DNA in the presence of alcohols. There is little difference in the spatial dependence associated with the exclusion of the alcohol methyl pentanediol (MPD) from Spermidine3+-DNA, Cobalt3+-DNA, or NaBr-DNA arrays. The hydration of phosphate groups on the DNA backbone likely dominates forces. Any additional contribution from electrostatics and a lowered solution dielectric constant is minimal. We are presently examining the interaction of 20 alcohols with highly charged Spd3+-DNA arrays in order to analyze the chemical features of alcohols that determine exclusion. To a surprisingly good first order approximation, exclusion amplitudes simply scale with the number of alkyl carbons without bound hydroxyl groups in the alcohol. In contrast, carbons with bound hydroxyl groups are practically invisible to DNA. We will use this same set of 20 alcohols to probe their exclusion from nonpolar hydroxypropylcellulose arrays. We anticipate that exclusion from this hydrophobically modified polysaccharide will scale with the number of polar hydroxyl groups in the alcohol. We have finished constructing a magnetic tweezers apparatus for single molecule force measuuurements. Our initial experimments will focus on measuring the free energy changes accompanying tightly packaging DNA using various divalent, trivalent, and higher valence cations, including naturally occurring protamines used in packaging DNA in the sperm heads of several organisms. As naturally found in the cell or virus, DNA is highly condensed. Our ultimate goal is to apply the lessons from direct force measurements to the recognition reactions that control cellular processes. We are measuring differences in water sequestered by complexes of several sequence specific DNA binding proteins with varying DNA sequences, with particular emphasis on correlating binding energy and water retained and on the energy necessary to remove this water from complexes. We previously observed that the osmotic sensitivity of the specific-nonspecific equilibrium binding constant of the restriction endonuclease EcoRI was only slightly dependent on the nature of solute used to set water activity. This led us to conclude that the extra water retained by the nonspecific complex is sequestered in a space sterically inaccessible to solutes likely at the DNA-protein interface. Since there is no structure of the nonspecific complex of EcoRI available, we could not directly confirm this conclusion. In order to validate the connection between osmotic stress measurements and structure, we are investigating the difference in sequestered water between specific and nonspecific complexes of a second restriction endonuclease, Bam HI. X-ray structures are available for both specific and nonspecific BamHI-DNA complexes. A comparison of the two structures shows that the nonspecific complex has lost the direct contacts of the protein with DNA bases in the major groove seen in the specific sequence complex. Instead a large cavity is seen between the major groove of the DNA and the recognition protein surface. The cavity is large enough to hold about 150 waters. Our initial kinetic and equilibrium competition experiments using the osmotic stress approach indicates the nonspecific BamHI ? DNA complex sequesters about 130 waters more than the specific complex of the enzyme, correlating well with the structural data. We are also investigating the correlation between water and binding strength of DNA complexes with lambda Cro repressor. This protein is responsible for the induction of the lytic phase of lambda phage. The recognition stringency for this repressor is more typical of specific sequence DNA-protein binding systems than the restriction nucleases we have investigated previously. Single base pair changes from the optimal recognition sequence only reduce binding energies by a few kT. We have constructed a series of DNA fragments with consensus and altered Cro recognition sequences that span a range of about 1000 in binding constant. Results for the osmotic pressure dependence of dissociation rates and of the competitive equilibrium binding constants for the various complexes indicate that as the binding energy decreases the number of sequestered waters increases. For about every factor 10 decrease in the binding constant an extra 7-10 water molecules are incorporated by the complex. The insensitivity of the number of extra water molecules sequestered by these complexes to solute identity indicates these waters are also likely at the DNA-protein interface, mediating suboptimal contacts between the two surfaces.